Bacteria of the genus Chlamydia are obligate intracellular parasites and include common human pathogens such as Chlamydia trachomatis, a leading cause of sexually transmitted diseases and blinding trachoma. Unlike most bacterial pathogens, C. trachomatis alternates between two physiologically distinct cell forms to establish infection: the infectious Elementary Body (EB) and replicative Reticulate Body (RB). Two histone related proteins, HctA and HctB, regulate compaction and relaxation of chlamydial chromatin during transition between developmental forms and control RNA polymerase access to the DNA. Biogenesis of the chlamydial replication niche depends on the re-initiation of protein expression as the EB germinates into the RB cell type. The factors controlling chlamydial gene expression during differentiation remains a significant knowledge gap. 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) is an intermediate of the methylerythritol phosphate (MEP) pathway for isoprenoid synthesis that promotes dissociation of chlamydial histones from isolated EB chromatin structures. The MEP pathway starts with condensation of the glycolytic intermediates glyceraldehyde 3-P and pyruvate and is therefore likely to be regulated by glycolytic activity. Expression of the chlamydial hexose phosphate transporter UhpC at the onset of morphological differentiation and recently demonstrated metabolic activation of EBs by glucose 6-P in vitro suggests that activation of glycolysis and pathogen energy metabolism is temporally linked to chromatin decondensation. We hypothesize that the histones, by binding to specific sites or structural elements on the chromosome, allow ordered control of early gene expression by affecting the access of regulatory proteins to promoters and genes involved in the control of differentiation. We further predict that initiation of metabolism is a key element in controlling histone release. The aim of this proposal is to determine the role of chromatin structure in developmental regulation and to establish a link between initiation of EB energy metabolism and regulation of EB chromatin structure immediately upon infection. The relationship between chromatin structure and de novo gene transcription will be determined using high throughput DNA- and RNA-seq techniques to measure changes in DNA accessibility and RNA transcription upon incubation of bacteria in a host cell-free medium containing glucose 6-P, and during early differentiation. Comparing the regulation of the EB chromatin structure early in development in vivo to chromatin structure changes of activated EBs in vitro will provide unprecedented insight into the mechanisms of EB germination. Additionally, as the MEP pathway is not present in mammalian cells, understanding the novel role of metabolism in activating the chlamydial pathogenic cycle will provide potential avenues for preventing or treating chlamydial infections.